Although there has been extensive work developing microfabrication and microfluidic technology for biomedical applications, there has still been limited progress in moving the technology broadly into biomedical research laboratories. Part of the issue is a lack of familiarity with the capabilities of microfabrication on the part of biomedical researchers. In addition, many potential research projects have constraints that require extensive customization and multiple design iterations, which may not be achievable with the limited number of commercial products available. In an effort to lower the barriers for applying microfabrication techniques to a wide range of biomedical problems, we have developed an in-house microfabrication capability that enables us to make single-layer templates for PDMS or hydrogel devices using a convenient dry-film resist process. Although the resolution, device yield, and complexity are somewhat lower than those achievable with a dedicated cleanroom, they are nonetheless sufficient for many experiments on cells. Furthermore, the instrumentation complexity, fabrication cost, and turnaround time are greatly reduced, enabling rapid cycling through design parameters as needed. This year, we continued to refine protocols for patterning two commercial dry-film resists. As a result, we are able to reliably pattern template features with lateral dimensions as small as 10 microns, and with heights ranging from 15 microns to a few hundred microns, on either flexible or rigid substrates. In addition, we have developed protocols for patterning several formulations of SU-8, a spin-on resist, which extends our capabilities down to layer thicknesses of a few microns and lateral resolution of 5 microns. We have also developed protocols for using these templates to generate microstructured PDMS, agarose, and PEGDA hydrogels. Finally, we continue to develop protocols for surface modification of PDMS and other polymers, including the irreversible bonding of PDMS to glass, using instrumentation in our laboratory, as well as techniques for connecting devices to flow-control instruments, such as syringe pumps. Through continued collaboration with scientists at NIST, we are also able to access the nanofabrication facilities at NIST to make more complex and higher-tolerance structures as needed. These capabilities have found application in a number of projects, representing a broad variety of interests and institutes, over this past year. Aside from the projects discussed below, we have trained researchers in basic microfabrication techniques, including representatives from other laboratories in NHLBI, NCI, and NIBIB. One longer-running project is an ongoing effort in collaboration with LSB, NIAID, to study chemotaxis in 3-D collagen matrices, for which we have been developing and refining a microfluidic agarose device compatible with high-resolution fluorescence and two-photon imaging. This year we implemented a mixing tee on a separate platform, which together with programmable syringe pump flow control has enabled the formation of reproducible time-varying gradients. In addition, we are in the process of developing a hybrid multilayer device capable of independently controlling the degree of collagen fiber alignment and the chemical gradient. A second project is the continuing development, in collaboration with LCE, NHLBI, of gratings for phase-contrast x-ray imaging, which involves the use of the NIST nanofabrication facilities as well as our own equipment. Over this past year, our group has contributed to refinement of the grating fabrication parameters as well as to the development of protocols for the replication, transfer, and functional characterization of gratings. A third ongoing project, in collaboration with LCMB, NCI, is an effort to confine B-T cell pairs in microwells such that the intercell junction is perpendicular to the optical axis, in order to enable high-speed, high-resolution imaging of synapse formation;this has been successfully implemented with PDMS, and we continue to investigate the use of other materials more closely index-matched to the culture media. In a fourth project we have been continuing the development, fabrication, and characterization of thin hybrid polymer films made by spin coating for use in operator-independent, high-resolution, laser capture microdissection. This work, begun as part of an inter-institute Director's Challenge project with researchers in NIMH, NCI, and NICHD, is aimed at developing methods for tissue-based capture of subcellular structures for mass spectrometry-based proteomic analysis. A fifth project, started this year, is the development and implementation of a PDMS microfluidic gradient generator for the deposition of chondroitin sulfate proteoglycans on substrates for neural cell culture, that we are using, in collaboration with DN, CBPC, NHLBI, to gain better understanding of the role these molecules play in axon growth and guidance. Finally, we are continuing work on a project aimed at miniaturizing the luciferase immunoprecipitation system (LIPS) assay developed in LSB, NIDCR, which uses a fusion protein consisting of Renilla luciferase and an antigen of interest to probe for antibodies in human serum. We have demonstrated successful function of the assay in a simple microfluidic format for diagnosis of HSV2 status in a panel of serum samples. Our ongoing work is focused on boosting the signal to a level that would enable the use of battery powered electronics for detection, and on incorporating passive flow controls into the device design in order to increase the assay automation.